Brain metastases, which occur in 30-40% of all cancer patients, are an important cause of cancer morbidity and mortality, and their frequency is rapidly increasing. Mechanisms responsible for malignant melanoma progression to highly aggressive brain-metastatic disease remain largely unknown. Main objective of our laboratory is to understand molecular determinants of brain-metastatic melanoma (BMM), and to use this knowledge for developing novel therapies to prevent brain metastasis. As the result of previous RO-1 funding and using our BMM cellular models, we have accumulated evidence demonstrating that the neurotrophin receptor p75NTR and neurotrophin-regulated heparanase (HPSE) are critical determinants of brain metastasis. HPSE is a metastasis - promoting enzyme which degrades the heparan sulfate glycosaminoglycan chains (HS) of HS proteoglycans (HSPG), which are ubiquitous components of the cell surface - extracellular matrix interface. In contrast, there is the recent notion that a different HS-degrading enzyme, bacterial heparinase-lll (Heplll), can inhibit the metastatic process. Importantly, HPSE studies have led us to novel investigations analyzing the differential HSPG degradation by heparanases - HPSE and Heplll - and how their action can modulate the BMM phenotype. Hypotheses for this application are: 1) HPSE modulates the BMM onset by degrading specific cell surface and ECM HSPG, 2) HS from HSPG, depending upon their selective degradation by heparanases, affect cell signaling and act as cryptic inhibitors or promoters of BMM, and 3) structural characteristics of HS, differentially cleaved by heparanases, correlate with melanoma progression to the brain-metastatic phenotype. The work proposed directly addresses issues raised by the N.C.I. Brain Tumor PRG report, in particular it relates to an improved understanding of mechanisms underlying the establishment and spread of brain metastasis. We will test these hypotheses by pursuing the following SPECIFIC AIMS: 1. To determine BMM modulation by inhibiting HSPG/HS expression and function. 2. To define effects of BMM/brain endothelial cell signaling by heparanases and heparanases- cleaved HS. 3. To determine the structural composition of HS cleaved by heparanases and their correlation with BMM. Relevance. Brain metastasis represents one of the most devastating aspects of cancer. Research outlined here will help in delineating molecular events associated with malignant melanoma growth and brain metastasis, as influenced by enzyme-driven interactions between tumor cells and extracellular matrix